Decoding the genetic structure of conjugative plasmids in international clones of Klebsiella pneumoniae: A deep dive into blaKPC, blaNDM, blaOXA-48, and blaGES genes

Carbapanem-resistant Klebsiella pneumoniae is a globally healthcare crisis. The distribution of plasmids carrying carbapenemase genes among K. pneumoniae poses a serious threat in clinical settings. Here, we characterized the genetic structure of plasmids harboring major carbapenemases (e.g. blaKPC, blaNDM, blaOXA-48-like, and blaGES) from K. pneumoniae using bioinformatics tools. The plasmids carrying at least one major carbapenemase gene were retrieved from the GenBank database. The DNA length, Inc type, and conjugal apparatus of these plasmids were detected. Additionally, allele types, co-existence, co-occurrence of carbapenemase genes, gene repetition, and sequence types of isolates, were characterized. There were 2254 plasmids harboring carbapenemase genes in the database. This study revealed that blaKPC-2, blaNDM-1, blaOXA-48, and blaGES-5 were the most prevalent allele types. Out of 1140 (50%) plasmids were potentially conjugative. IncFII, IncR, IncX3, and IncL replicon types were predominant. The co-existence analysis revealed that the most prevalent of other resistance genes were blaTEM-1 (related to blaKPC), blaOXA-232 (related to blaOXA-48), bleMBL (related to blaNDM), and aac (6′)-Ib4 (related to blaGES). The co-occurrence of carbapenemases was detected in 42 plasmids while 15 plasmids contained carbapenemase gene repetitions. Sequence alignments highlighted that plasmids carrying blaKPC and blaOXA-48-like were more homogeneous whereas the plasmids carrying blaNDM were divergent. It seems that K. pneumoniae utilizes diversity of genetic flexibility and recombination for resistance against carbapenems. The genetic structure of the plasmids showed that class I and III, Tn3 family, Tn5403 family derivatives, and Tn7-like elements were strongly associated with carbapenemases. The mobilizable plasmids carrying carbapenemases play an important role in the spread of these genes. In addition, gene repetition maybe is related to carbapenem heteroresistance. According to MST (minimum spanning tree) results, the majority of plasmids belonged to sequence type (ST) 11, ST14, and ST12. These international clones have a high capacity to acquire the carbapenemase-containing plasmids.


Introduction
Klebsiella pneumoniae is one of the most important opportunistic pathogens found in nosocomial and community-acquired infections as well as in asymptomatic fecal carriages [1,2].K. pneumoniae isolates play an important role in causing serious infections, including pneumonia, bloodstream infections, urinary tract infections, surgical site, and burn wound infections [3].In addition, the presence and spread of resistance genes pose a challenge to successful treatment.Extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase-producing K. pneumoniae (CPK) isolates are repeatedly associated with failure of antibiotic therapy [4].The mortality rate caused by carbapenem-resistant K. pneumoniae (CRK) isolates is significantly twice as high compared with infections caused by carbapenem-susceptible isolates [5].Several reasons, including severe co-morbidities, higher virulence of CRK isolates, improper use of antibiotics along with high level of toxicity, are associated with the increase in mortality rates [6].
Various classes of carbapenemase, according to Ambler classification, are detected in K. pneumoniae isolates, including class B, metallo-beta-lactamases (New Delhi Metallo-beta-lactamase-NDM), class D carbapenemases (e.g.OXA-48), and class A carbapenemase of K. pneumoniae (e.g.KPC) [7].Although CPK isolates are common in different regions, KPC is endemic in the United States and some European countries, including Greece and Italy.Whereas, MBLs (metallo-beta-lactamases, including NDM-1) and OXA-48-like are found mainly in Asian countries such as Turkey, India, Pakistan, and the Middle East region [8].It seems that the presence of carbapenem resistance genes on conjugative plasmids could lead to their worldwide dissemination and therefore the high prevalence of CPK isolates could be a serious problem for the health care system all over the world.Also, the coexistence of carbapenemase-encoding genes with other resistance genes such as aminoglycoside-modifying genes in K. pneumoniae isolates exacerbates the problem of antibiotic resistance as a challenge in the curing of infectious [9,10].The harboring of these resistance genes on mobile genetic elements (MGEs), including class 1 integron, transposons, and insertion sequences are usually carried on conjugative plasmids, contribute to expansion of antimicrobial resistance (AMR).
Resistance genes are usually associated with specific clonal groups.Strains of multidrugresistant K. pneumoniae isolates are generally found in sequence type (ST) 147, ST15, and ST258.In addition, virulence genes are also carried in several specific STs, including ST147, ST15, ST48, ST101, and ST383.Plasmids harboring carbapenem-resistance genes, including bla KPC belonging to IncFIIk/IncR, are typically found in ST11 and play an important role in the spread of resistance genes in Asian countries, including China [11].Moreover, ST11 is highly related to hypermucoviscous isolates of K. pneumoniae.Isolates of carbapenem-resistant K. pneumoniae ST11 can acquire the virulence of large plasmids from hypervirulent isolates [12].In other words, virulence plasmids from hypervirulent K. pneumoniae isolates can be transferred to resistant isolates [13] and Therefore, the spread of these high virulence and resistance capacity plasmids among prevalent STs such as ST11 could be complicated and potentially life-threatening for patients, especially those hospitalized in intensive care units (ICUs) for a long time [14].
According to resistant K. pneumoniae, various epidemiological studies have been conducted so far.However, it is still important to analysis that led to the decipher of the genetic structures of plasmids harboring AMR genes.Also the investigation of the MGEs associated with the resistant plasmids and prevalent STs, play an important role in the spread of antibiotic resistance among bacteria.Characterization of these MGEs can give rise to a better insight of how antimicrobial resistance might widely spread.In this study, we detected and compared the different allele types of the major carbapenemase genes, including bla KPC , bla NDM , bla OXA- 48, and bla GES from K. pneumoniae using bioinformatics tools.In addition, we characterized the genetic properties of carbapenemases harboring plasmids including replicon types, conjugation ability, the co-existence (e.g.linkage of carbapenemases with other antimicrobial resistance genes), co-occurrence (e.g.having at least two carbapenemase genes in one strain), gene repetition, alignment and phylogenetic relatedness.

Allele types determination of carbapenemase genes
The allele types of the mentioned carbapenemase genes located on the completed plasmids and partial DNA fragments were detected using the beta-lactamase database (http://bldb.eu/).The criteria were 100% identity and 100% coverage.In addition, the prevalence of each allele type was calculated.

Detection of other AMR genes on retrieved DNAs
The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/home) was used to detect the presence of antimicrobial resistance genes against carbapenem, extended-spectrum beta-lactams, fluoroquinolones, aminoglycosides, chloramphenicol, tetracycline, macrolide, and other antibiotics [15].The co-existence (gene linkage) of other antimicrobial resistance genes with the major carbapenemase genes in the plasmids was characterized.Moreover, the availability of the at least two major carbapenemase genes in an isolate were considered as co-occurrence.

Clonal relatedness of strains harboring carbapenemase genes
The distribution of the major carbapenemase-encoding genes among the different STs was assessed.For each plasmid, the ST of the associated chromosome was determined using seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) via the PubMLST database (https://pubmlst.org/)[19].The clonal relatedness of STs was characterized using PHYLOViZ version 2.0 to generate a minimum spanning tree (MST) for all STs [20].

The distributions and the allele types of the carbapenemase genes
Two thousand two hundred and fifty-four (2254), including 1132 plasmids harboring bla KPC, 495 plasmids containing bla NDM, 617 plasmids with bla OXA-48 -like , and 10 plasmids with bla GES were retrieved from GenBank database in microbial BLAST.In addition, 362, 132, 124, and 11 DNA partial fragments carrying bla KPC, bla NDM, bla OXA-48 -like , and bla GES, respectively were found according to the standard BLAST.Moreover, all data on the geographical regions, isolation sources, years and hosts of harboring crabapenemases of isolates have been shown in Table 1 and S1 File.

The co-existence of other antimicrobial resistance genes in the plasmids
Different antimicrobial resistance genes against various classes of antibiotics, including extended-spectrum beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicol, tetracycline, macrolide, fosfomycin and sulfonamides along with genes encoding efflux pump proteins and antiseptic-resistance genes, were found in plasmids carrying a least one carbapenemase gene.The most prevalent co-existed genes in plasmids harboring bla KPC , bla OXA , bla NDM , and bla GES were bla TEM-1 , bla OXA-232 , ble MBL, and aac (6 0 )-Ib4, respectively.See

The co-occurrence of carbapenemase genes
Analysis of the data retrieved from the GenBank database revealed that the co-occurrence of carbapenemase genes in different plasmids but in the same strains.This coincidence was found in forty-two genomes.The co-occurrence of carbapenemase genes with predominant allele types was as follows.A number of seventeen plasmids had bla NDM-1 and bla KPC-2 .Four plasmids had bla OXA-48 /bla NDM-1, and three plasmids had bla OXA-48 /bla KPC-2 .The rest of the plasmids having the co-occurrence genes has been shown in Table 2. Also four plasmids, including NZ_CP094991, NZ_CP104796.1,NZ_CP086664.1, and NZ_CP090126.1 simultaneously contained three carbapenemase genes, including bla OXA-48 /bla NDM-1 /bla KPC-2, bla OXA- 181 /bla NDM-1 /bla NDM-4, bla OXA-48 /bla NDM-1 /bla KPC-2, and bla NDM-1 /bla KPC-2 /bla KPC-2 , respectively.See Table 2.The conjugal plasmids were various among the strains.For example, in NZ_CP050376.1 and NZ_CP041082.1 the plasmids were potentially conjugative, whereas in some other strains, e.g.CP065949.1 and NZ_CP024038.1 one plasmid was potentially conjugative and another plasmid was not conjugative.In strains with plasmids containing three carbapenemase genes, the plasmids were potentially conjugative or at least were mobilizable (only lacked oriT).

The gene repetition in the plasmids
According to this study, there were 15 plasmids with carbapenemase gene repetitions.See Table 3.In eleven plasmids harboring bla   been detected.No repetition was found in plasmids carrying bla OXA-48 .The plasmids containing bla KPC with two or three copy numbers mostly belonged to ST11.They had IncFII replicon type and was potentially conjugative.While plasmids with bla NDM-1 were associated with ST2816 and ST15, had mostly IncFIB, and were potentially conjugative or mobilizable.Both plasmids harboring bla GES-24 belonged to ST12, IncFII, and IncCol of plasmids and they didn't have conjugal systems.
The bla NDM-1 gene was found between IS30 and Tn3-like transposase genes.Mapping of this transposon revealed that ble MBL , tat, cutA, groES, and groEL were located adjacent to bla NDM-1.The bla OXA-48 was located between IS1-like and IS4-like families and lysR was also a neighbor.Mapping of bla KPC-2 showed that this carbapenemase gene was flanked by the transposase family ISKpn27 and transposase family ISKpn6.Class 1 integron and class 3 integron were found in plasmids containing bla GES-5 and bla GES-24 , respectively.Other genes, including      aac ( 6)-Ia, cat, ant, DUF86, invA, and blaA were found adjacent to bla GES-24 and dfrB1, bla OXA-10, aac(6')-Ib4 and qacE delta1 were seen near to bla GES-5 .The highly prevalent genetic structures associated with the carbapenemase genes have been shown in Fig 4.

Plasmid analysis
The sequence comparisons showed that plasmids carrying bla KPC and bla OXA-48 appear to be more homogeneous and conserved, whereas the plasmids carrying bla NDM were more heterogenic and distributed in the circular dendrogram.See Fig 5.
The plasmids harboring bla KPC which were located in the same cluster mostly had IncFII replicon types and were mainly potentially conjugative.In addition, the plasmids with bla OXA- 232 had the ColKP3 replicon type and were all non-conjugative, whereas bla OXA -48 had the IncL replicon type and were all potentially conjugative.Among plasmids harboring bla NDM , the conjugation pattern and Inc type were different.Plasmids in the same cluster had the same replicon type (e.g., IncC) but a different Inc type in comparison to plasmids that were in a different cluster (which had IncFIB replicon type).It should also be noted that some of these plasmids that were in one cluster, were all mobilized.While the others in the same cluster had a different conjugal pattern.

Discussion
Carbapenem-resistant K. pneumoniae is one of the most challengeable causes of communityacquired and nosocomial infections which can increase morbidity and mortality rate [21].Multidrug-resistant K. pneumoniae isolates complicate treatment, and carbapenems are one of the last-line agents to combat these infections.Therefore, carbapenem resistance could make the situation worse [22].The presence of carbapenemase genes on conjugative plasmids also contributes to the higher dissemination rates [12].According to the latest report of CDC and WHO K. pneumoniae is considered as urgent priority and the presence of resistance genes, including bla KPC2 and bla KPC3 along with bla NDM and bla OXA-48-like as the most prevalent carbapenemase genes could be a worrying issue in public health [23].In the current study, bioinformatics tools were used to obtain more information about the genetic characteristics of K. pneumoniae plasmids harboring carbapenemase genes.
ST11 is one of the most common ST in K. pneumoniae isolates.Recently, outbreaks of ST11 carbapenem-resistant hypervirulent K. pneumoniae have been reported in Asian countries, such as China, and ST11 also accounts for 12% of carbapenem-resistant K. pneumoniae in Europe.Apart from the wide distribution of ST11 and its isolation from human samples, ST11 could also be isolated from non-human environments, according to Campos-Madueno et al [24].ST11 is a single-locus variant of ST258, is one of the most common member of CG258, and is more distributed compared to ST258 and ST512 [25][26][27].The presence of ESBL encoding genes, including bla CTX-M-65 and bla TEM-1 , which were also seen in this study, is prevalent in ST11 [26].According to the current study, plasmids harboring bla KPC and bla NDM belonging to ST11.Plasmids with bla KPC , especially bla KPC-2 , mostly had IncFII and IncR replicon types and 39.6% were potentially conjugative.Plasmids with bla NDM had IncX3 and IncC replicon types and 54.5% were potentially conjugative.IncFII is one of the most prevalent plasmids found in carbapenem-resistant isolates and it is restricted to the Enterobacteriaceae family [28].IncX3 is also found mainly in Enterobacteriaceae, has high transmissibility, and facilitates the spread of bla NDM among K. pneumonae [29,30].Regarding the current data, the conjugative plasmids harboring carbapenemase and ESBL genes belonging to ST11 could be notable as they could affect the dissemination of resistance genes.On the other hand, due to the genetic structure, bla NDM-1 was accompanied by other resistance genes, including tat and ble MBL, localized on the Tn3-like elements.The Tn3 family is one of the most important mobile genetic elements with the ability to spread a variety of passenger genes, including those conferring resistance to several classes of antibiotics, including carbapenem and colistin resistance [31].Mapping of the structure containing bla KPC also revealed ISKpn6 and ISKpn27 elements.The core structure of ISKpn27/ISKpn7-dnaA-bla KPC-2 -ISKpn6 is highly epidemic in KPC-producing K. pneumoniae isolates [32].The presence of insertion sequences and transposons with high transmission capacity harboring carbapenemase genes in plasmids associated with ST11 K. pneumoniae isolates could render these isolates be lethal and cause life-threatening infections.
In the current study, ST14 was another predominant sequence type associated with plasmids containing bla OXA-48.ST14 is one of the major STs carrying multiple resistance genes and is common and associated with pediatric and neonatal infections [33].Available studies indicate that ST14 K. pneumoniae isolates can lead to fatal infections.According to this study, plasmids containing OXA gene (e.g., bla OXA-48 ) and associated with ST14 mostly had IncL replicon type and were conjugative.In addition, mapping of the construct of the cassette containing OXA revealed that this gene is flanked by IS1 and IS4 families.Insertion of these elements into K. pneumoniae isolates with notable virulence factors, including ompK36 results in higher resistance to carbapenems and an increase in the probability of the treatment failures.On the other hand, the adjacency of bla OXA-48 with lysR can worsen the situation since this gene plays an important role in mediating antibiotic resistance and increasing the virulence of K. pneumoniae isolates [34].In this study, ST12 is another predominant ST associated with plasmids carrying bla GES-24 with a notable genetic map.The co-existence of bla GES-24 with resistance genes, including aac (6)-Ia, cat, and blaA genes leads to high resistance to multiple antibiotic classes, maybe increase the rate of treatment failure.
One of the most outstanding findings of the current study is the multi-harbor STs.ST101, ST147, and ST16 are three important STs involved in the spread of K. pneumoniae isolates.Overall, the data from the current study showed that the plasmids associated with ST147 and ST101 had mostly plasmid with IncL (with bla OXA ) and IncFIB and IncFII replicon types (containing bla KPC and bla NDM ), that were almost potentially conjugative or at least mobilizable.The predominant alleles were bla OXA-48, bla KPC-2, and bla NDM-1, however, bla OXA-10, bla OXA- 181, bla OXA-232, bla NDM-5, bla NDM-7, bla NDM-29 and bla NDM-9 could also be found in plasmids associated with ST147.These STs are highly associated with lethal infections that according to available studies ST101 could be assumed as a global threat since it plays a major role in the dissemination of resistance genes, including colistin-resistance [35].ST16 is also a major clone associated with the spread of bla NDM-1 and bla OXA232 and is thought to be one of the most widespread and high-risk clones worldwide [36].The results of our study also confirmed these data; however, evaluation of the plasmids examined revealed that ST16 might be associated with plasmids containing bla NDM-4 and bla NDM-5 , bla OXA-48 , and bla OXA181 .In addition, according to this study, ST16 was associated with IncFII, IncFIA/B, IncL, and Inc ColKP3.Shukla et al, also reported that ST16 carried mainly ColKP3 [37].Importantly, IncFII, IncFIA/ B, and IncL were mostly conjugative or mobilizable.Whereas ColKP3 was mostly non-conjugative.The presence of non-conjugative plasmids in K. pneumoniae ST16 may be a noteworthy clue affecting the distribution of carbapenem-resistance genes.Taken together, the presence of these STs with high transmission capacity and multiple carbapenemase genes is a big concern in public health.
Our study also revealed remarkable points regarding the co-existence and co-occurrence of resistance genes.The bla TEM-1 , bla OXA-232 , ble MBL , and aac(6')-Ib4 were the most abundant genes in the same plasmids harboring the carbapenem resistance genes.Several genes responsible for resistance to different classes of antibiotics, including aminoglycosides, extendedspectrum beta-lactamase, and carbapenem, were found to coexist with bla KPC , bla OXA-48 , bla NDM , and bla GES .The co-existence of ble MBL and bla NDM was predictable, as ble MBL is always downstream of bla NDM [38].The presence of ble MBL responsible for bleomycin resistance (as a glycopeptide antibiotic) with bla NDM could increase the rate of treatment failure.In addition, the presence of two or three plasmids with carbapenem resistance genes among different strains seems to be a critical point.Especially because these plasmids were mainly potentially conjugative and carried transposons such as the Tn3 family and integron class I. Tn3 is one of the most widespread transposase families responsible for the spread of resistance genes [31].Therefore, these strains with different plasmids, each containing a different resistance gene.Plasmid analysis revealed a high similarity between plasmids containing bla KPC-2 and bla OXA-48 , while bla NDM seems to be heterogeneous.This could be a remarkable point as it shows that bla NDM could be placed in different plasmids with different sequences, which would worsen the situation of antimicrobial resistance.

Conclusion
The co-existence of different classes of resistance genes, co-occurrence of various carbapenemase genes on separate plasmids, and gene repetition in a plasmid were notable findings of this study.Assessment of genetic characteristics of the plasmids also revealed that bla NDM -harboring heterogenic plasmids had a high capacity for dissemination.multi-harbor carbapenemases STs could highly affect the exacerbation of the antimicrobial resistance in this bacterium.K. pneumoniae appears to employ multiple genetic strategies for resistance against carbapenem antibiotics.First, gene repetition and locating carbapenemase genes associated with class 1 and 3 integrons, ISKpn and Tn3 plays important roles in DNA recombination.In addition, the placement of these DNA fragments on transferable plasmids paves the way for widespread expansion of antimicrobial resistance.Finally, the successful and international clones (ST11, ST14, ST437, ST23, ST307, ST101, ST147, ST16, ST17, ST35 and ST37) with high ability to capture carbapenemase-containing plasmids are actually the last circle of this journey which has dramatically increased antibiotic resistance all over the world.It seems that K. pneumoniae is collecting various resistance genes and virulence factors with all its genome capacity.Such genetic flexibility of a superbug is not only astonishing but also a very serious health threat.In the future, new methods (e.g.vaccination, novel drug targets and antibiotics, and new combination therapy such as antibodies and antibiotics) should be used to fight against K. pneumoniae.

Fig 1 .
Fig 1.The flowchart conducted in the current study.Two BLAST approaches including microbial BLAST and standard BLAST had been applied to retrieve all completed plasmids and DNA fragments carrying carbapenemase genes.All tools and functions have been shown in this pipeline.https://doi.org/10.1371/journal.pone.0292288.g001 Fig 3 and S3 File.

Fig 2 .
Fig 2. The prevalence of allele types of the major carbapenemase genes.A) The different of allele types of bla KPC gene, B) The proportions of all allele types observed in bla NDM gene, C) The frequency of bla OXA-48 -like allele types, D) All allele types detected in bla GES gene.https://doi.org/10.1371/journal.pone.0292288.g002

Fig 4 .
Fig 4. The genetic environments of major carbapenemase genes associated with class 1 integrons, insertion sequences and transposons.A) The genetic features of bla NDM-1 flanked by Tn3 like elements.The bla NDM-1 comes together by other resistance genes, including tat and ble MBL, localized on the Tn3-like elements.B) The genetic environment of bla OXA-48 is between IS1-like and IS4-like families and lysR is also a neighbor.C) The bla KPC-2 flanks between ISKpn6 and ISKpn27.The core structure of ISKpn27/ISKpn7-dnaA-bla KPC-2 -ISKpn6 is highly epidemic in KPC-producing K. pneumoniae isolates.D) The bla GES-5 associated with intI1, E) While genetic features of bla GES-24 related to intI3.https://doi.org/10.1371/journal.pone.0292288.g004

Fig 5 .
Fig 5.The circular sequence alignment of eighty-five plasmids harboring multiple major carbapenemase genes.Data shows that plasmids carrying bla KPC are more convergent.On the hand, bla NDM encoding plasmids are divergent and have different genetic characteristics (molecular weight, replicon typing, conjugal apparatus and other antimicrobial genes).https://doi.org/10.1371/journal.pone.0292288.g005

Table 3 . Gene repetition of carbapenemases which has been found from the plasmids.
*Gene duplication was detected in DNA sequence with point mutations and frameshifts.https://doi.org/10.1371/journal.pone.0292288.t003